DNA fingerprinting or DNA typing involves the display of a set of DNA fragments from a selected DNA sample. In almost all living organisms with the exception of viruses, restriction digests of the total genomic DNA of the organism yield so many bands that it is not possible to score individual bands. Accordingly, fingerprinting methods are based on the principle that only a small fraction of the DNA fragments are visualized so as to yield a simple banding pattern which makes up the DNA fingerprint. A variety of techniques for DNA fingerprinting are currently available.
Until recently, the most widely used method for DNA fingerprinting was Southern hybridization. This procedure requires digesting the DNA of the organism with restriction endonuclease, fractionating the restriction fragments by gel electrophoresis, transferring and binding the fractionated DNA fragments onto membranes and hybridizing the membrane with a specific DNA fragment or probe. The probe forms a double-stranded DNA molecule with the DNA fragment or fragments on the membrane having complementary nucleotide sequences. The probe is typically tagged with a visible marker for easy detection of the DNA fragment. DNA polymorphisms, and more specifically restriction fragment length polymorphisms, are identified by differences in size of the corresponding restriction fragments to which the probe attaches. However, Southern hybridization is both laborious and time consuming.
Accordingly, a number of DNA fingerprinting techniques have been developed which use polymerase chain reaction (PCR) for detection of fragments. The selection of the fingerprinting technique to use is dependent upon the application, i.e., DNA marker typing, DNA typing, and the organism and size of the genome of the organism. Preferably, the fingerprinting technique requires no prior sequence analysis, primer synthesis or characterization of DNA probes. A number of fingerprinting techniques which meet these criteria have been developed. These include random amplified polymorphic DNA (RAPD; Williams et al. Nucleic Acid research 1990 18:6531-6535), DNA amplification fingerprinting (DAF; Caetano-Anolles et al. Bio/Technology 1991 9:553-557) and arbitrarily primed PCR (AP-PCR; Welsh, J. And McClelland, M. Nucleic Acid Research 1990 18:7213-7218). However, these PCR based fingerprinting methods are very sensitive to reaction conditions, DNA quality and PCR temperature profiles, thus limiting their use.
Amplified fragment length polymorphisms (AFLP) is a newer method for DNA fingerprinting involving application of PCR to amplify one or more restriction fragments from complex mixtures of DNA fragments obtained by digesting a genomic DNA molecule with restriction endonuclease. The general method for AFLP is disclosed by Zabeau and Vos in PCT Application WO 93/06239. In this method, primers used for amplification are not directed against a known DNA sequence but rather are designed such that they recognize the ends of the restriction fragments. The PCR primers taught by Zabeau and Vos comprise a constant nucleotide sequence part and a variable nucleotide sequence part. The constant sequence part of the nucleotide sequence is designed so that the primer matches with the base pairs of one of the DNA strands at the end of the restriction fragment. The variable sequence part designates a sequence consisting of selected nucleotides forming a sequence which then remains constant during amplification of a subset of restriction fragments and directs that preselection of tagged restriction fragments to be amplified in the PCR step. Selection is determined by the number of nucleotides residing in the variable length part of the primer. These are referred to as selective bases or selective nucleotides. Selectivity of the primer is suggested to theoretically increase with the number of selective nucleotides in the variable sequence part.
A number of examples of DNA fingerprinting via AFLP are disclosed in PCT Application WO 93/06239 with primers having a constant nucleotide part which matches the base pairs of various restriction fragments of cutting enzymes including PstI, MseI, EcoRI, Taq, AseI and Sse8387-I and variable regions with 1 to 3 selective nucleotides. Also disclosed is a primer set with a constant nucleotide part which matches the restriction fragment of the rare cutting enzyme, PstI, and variable regions with 5 selective nucleotides. Rare cutting enzymes are characterized by having a restriction site of 6 or more nucleotides.
More recent studies by Vos et al. (Nucleic Acid Research 1995 23(21) :4407-4414), however, indicate that primers for frequent cutter enzymes, e.g., MseI, lose selectivity if more than 3 selective nucleotides are incorporated into the variable region. Vos et al. teach a loss of selectivity with primers having four selective bases illustrated by numerous bands not detected in the corresponding fingerprints with primers having three selective bases. This is suggested to be indicative of the tolerance of mismatches in the amplification fragments using AFLP primers with four-base extensions.
In the present invention AFLP primers for frequent enzyme cutters which contain four or more selective nucleotides are provided which are able to maintain their selectivity while reducing complexity and increasing specificity in marker genotyping. In these primers, one or more nucleotides of the constant region of the AFLP primer are truncated as additional selective nucleotides are added to the variable region of the AFLP primer so that the length of the primer remains relatively the same. These AFLP markers are particularly useful in marker genotyping of large genomes. DNA markers for volume growth in loblolly pine have now been identified using AFLP primers of the present invention.